The first human clinical trial has been performed to assess the safety of recombinant adeno-associated virus (rAAV) vector-based gene delivery to the retina of patients with blindness from Leber congenital amaurosis (LCA) and mutations in the RPE65 (retinal pigment epithelium-specific protein 65-kDa) gene. Vision was safely restored in RPE65-LCA, a previously untreatable and incurable human retinal degenerative disease. At the end of the current grant period, we will have treated 15 patients representing two age groups: 8-17 (n=7) and >18 years (n=8), using 4 different doses and two approaches: a single injection site in one eye (Cohorts 1-3, n=9) and two injection sites in one eye at the same surgical session (Cohorts 4 and 5, n=6). Five specific aims are now proposed to complete this early phase trial. Aim 1- Evaluate the long-term safety of subretinal rAAV2-hRPE65 gene therapy in all 15 subjects with the FDA-mandated three years of follow- up. Aim 2- Evaluate the efficacy of gene therapy to restore vision by determining: a) if the early improvement in vision persists by 3 years post-treatment; b) whether pre-treatment photoreceptor-RPE structure predicts the improvement in visual sensitivity in the post-treatment period; and c) if there are visual changes that emerge over a longer time course following treatment. Aim 3- Evaluate whether gene therapy alters the natural history of retinal degeneration by determining: a) the natural history of retinal degeneration in human RPE65-LCA in the cone-only fovea and in rod-dominated extrafoveal retina; and b) if treated retinal regions have a different natural history of photoreceptor loss. Aim 4- Study the properties of the restored visual cycle in treated patients using dark adaptometry and determine: a) if there is a dose-response function for the adaptation defect; b) the resulting rate kinetics for rods and cones as a function of remaining photoreceptor architecture and retinal location, pre-treatment visual function and the extent of improvement post-treatment; and c) if the dark adaptation defect changes with time after treatment. Aim 5- Assess whether mobility performance is affected by the gene therapy intervention and what quantified visual parameters make a difference in mobility. Answers will thus be sought to the many scientific and medical questions that have arisen from the procedures in these 15 patients. These answers should refine the approach and clarify expectations of gene therapy in this disease and lead to better approaches to other human genetic retinal diseases that are queuing up as future candidates for this type of therapy. RELEVANCE: The current proposal dovetails with the ongoing clinical trial. Many unanswered questions remain about the detailed results of this first human gene therapy for a genetic retinal disease. The five aims of this grant are both regulatory and scientific and will seek answers to questions critical to further development of this clinical trial and future gene therapy trials of retinal degenerative disorders.